"Apoptosis" refers to cell suicide that proceeds by an active, physiological process (Kerr, J. F., et al., Br. J. Cancer 26:239-257 (1972); Wyllie, A. H., Nature 284:555-556 (1980)). Cells that die by apoptosis undergo characteristic morphological changes, including cell shrinkage, and nuclear condensation and fragmentation. Apoptosis plays an important role in developmental processes, including morphogenesis, maturation of the immune system, and tissue homeostasis whereby cell numbers are limited in tissues that are continually renewed by cell division (Ellis, R. E., et al., Annu. Rev. Cell. Biol. 7:663-698 (1991); Oppenheim, R. W., et al., Neurosci. 14:453-501 (1991); Cohen, J. J., et al., Annu. Rev. Immunol. 10:267-293 (1992) M.C., Nature 356:397-400 (1992)). Apoptosis is an important cellular safeguard against tumorigenesis (Williams, G. T., Cell 65:1097-1098 (1991); Lane, D. P. Nature 362:786-787 (1993)). Defects in the apoptotic pathway may contribute to the onset or progression of malignancies. Under certain conditions, cells undergo apoptosis in response to forced expression of oncogenes, or other genes that drive cell proliferation; (Askew, D., et al., Oncogene 6:195-1922 (1991); Evan, G. I., et al., Cell 69:119-128 (1992); Rao, L., et al., Proc. Natl. Acad. Sci. USA 89:7742-7746 (1992); Smeyne, R. J., et al., Nature 363:166-169 (1993)). A variety of degenerative disorders may involve aberrant apoptosis, resulting in premature or inappropriate cell death (Barr, P. J., et al., Biotechnology 12:487-493 (1994)). Productive infection by certain viruses may depend on suppression of host cell death by anti-apoptotic viral gene products (Rao, L., et al., Proc. Natl. Acad. Sci. USA 89:7742-7746 (1992); Ray, C. A., et al., Cell 69:597-604 (1992); White, E., et al., Mol. Cell. Biol. 12:2570-2580 (1992); Vaux, D. L., et al., Cell 76:777-779 (1994), and inhibition of apoptosis can alter the course (i.e. lytic vs. latent) of viral infection; (Levine, B., et al., Nature 361:739-742 (1993)). Widespread apoptosis of T lymphocytes triggered by HIV infection may, at least in part, be responsible for the immune system failure associated with AIDS (Gougeon M., et al., Science 260:1269-1270 (1993)). The roles of apoptosis in normal and pathological cell cycle events are reviewed in Holbrook, N. J., et al., Eds., Cellular Aging and Cell Death, Wiley-Liss, Inc., Publisher, New York, N.Y. (1996).
The bcl-2 gene product has been intensively studied as a potent suppressor of apoptotic cell death. The bcl-2 gene was originally identified at the t(14:18) translocation breakpoint that occurs frequently in human B cell follicular lymphomas (Bakhshi, A., et al., Cell 41:899-906 (1985); Cleary, M. L., et al., Proc. Natl. Acad. Sci. USA 82: (1985); Tsujimoto, Y., et al., Science 229:1390-1393 (1985)). This translocation results in the constitutive activation of bcl-2 gene expression due to juxtaposition with the immunoglobulin heavy chain locus. Bcl-2 functions as an oncogene in this disease by inappropriately suppressing apoptosis that would normally limit the accumulation of these cells (McDonnell, T. J., et al., Cell 57:79-88 (1989); Hockenbery, D., et al., Nature 348:334-336 (1990)). Consequently, B cells accumulate during the indolent stage of the lymphoma due to their failure to die rather than by uncontrolled proliferation.
The anti-apoptotic activity of Bcl-2 is not restricted to B cells. A large number of studies have demonstrated that ectopic Bcl-2 expression can suppress apoptosis triggered by diverse stimuli in a multitude of cell lineages (Vaux, D. L., et al., Nature 335:440-442 (1988); Sentman, C. L., et al., Cell 67:879-888 (199 1); Strasser, A., et al., Cell 67:889-899 (199 1); Hockenbery, D. M., et al., Cell 75:241-251 (1993)). Bcl-2 blocks cell death induced by growth factor withdrawal, DNA damage, oncogene expression, oxidative stress, and viral infection. The ability of Bcl-2 to block apoptosis in virtually every system suggests that Bcl-2 is closely connected with the machinery that actually carries out the death program. This view is further supported by the conservation of Bcl-2 function across species. The ced9 gene in the nematode C. elegans functions to suppress programmed death in certain cell lineages of the developing worm (Ellis, H. M., et al., Cell 44:817-829 (1986)). Ced9 appears to be a functional homologue of Bcl-2, since Bcl-2 can complement ced9 in transgenic worms (Vaux, D. L., et al., Science 258:1955-1957 (1991)). Bcl-2 can also function in insect cells as demonstrated by the ability of Bcl-2 to suppress apoptosis induced by Baculovirus infection (Alnemri, E. S., et al., Proc. Natl. Acad. Sci. USA 89:7295-7299 (1992)). The molecular mechanism whereby Bcl-2 operates to block cell death is poorly understood.
Additional cellular genes that exhibit significant sequence homology with Bcl-2 have been identified and, where tested, these genes appear also to function as regulators of apoptotic cell death. One Bcl-2 relative, Bcl-X, was isolated by low stringency DNA hybridization to the Bcl-2 gene (Boise, L. H., et al., Cell 74:597-608 (1993)). The Bcl-X RNA is differentially spliced to produce a long form, termed Bcl-X.sub.L, and a shorter form, Bcl-X.sub.S bearing a short internal deletion. Bcl-X.sub.L functions to suppress cell death, much like Bcl-2, whereas the deleted form, Bcl-X.sub.S, can inhibit protection by Bcl-2 and may function as a "dominant negative" species. A second Bcl-2 relative, Bax, was identified biochemically as protein found in co-immunoprecipitates with Bcl-2 (Oltvai, Z. N., et al., Cell 74:609-619 (1993)). Isolation of the corresponding cDNA revealed that the Bax protein shows substantial sequence homology to Bcl-2. Bax forms heterodimers with Bcl-2 and appears to induce apoptosis and function as a negative regulator of Bcl-2 function. Ectopic expression of Bax was shown to block the protection against apoptosis afforded by Bcl-2 expression.
Two additional cellular Bcl-2 relatives, Mcl-1 and A1 (Kozopas, K. M., et al., Proc. Natl. Acad Sci. USA 90:3516-3520 (1993); Lin, E. Y., et al., J. Immunol. 151:1979-1988 (1993)) were originally isolated as mRNAs induced in response to specific stimuli: phorbol ester induced differentiation of myeloid leukemia cells (Mcl-1); and GM-CSF treatment of murine bone marrow cells (A1). It is not yet known whether either Mcl-1 or A1 can modulate apoptosis.
In addition to these cellular Bcl-2 relatives, a number of Bcl-2 homologues encoded by DNA viruses have been identified. The Epstein-Barr virus BHRF-1 gene product was noted to contain sequence homology to Bcl-2 and has subsequently been shown to function as a suppressor of apoptosis (Henderson, et al., Proc. Natl. Acad. Sci. USA 90:8479-8488 (1993)). Likewise, the African swine fever virus LMW5-HL gene encodes a protein structurally similar to Bcl-2 (Neilan, J. G., et al., J. Virol. 67:4391-4394 (1993)). The Adenovirus E1b 19kD protein appears to be functionally equivalent to Bcl-2, although the primary sequence homology is quite limited (White, E., et al., Mol. Cell. Biol. 12:2570-2580 (1992)). It is likely that these genes function to ensure replication of viral DNA by preventing apoptosis of the infected cell. The finding that unrelated DNA viruses have evolved genes that apparently function to mimic the action of Bcl-2, supports the conclusion that Bcl-2 represents an important apoptosis regulator.
The isolation and characterization of a bcl-2 related gene, termed bak, is described in co-pending U.S. application Ser. No. 08/321,071, filed Oct. 11, 1994, now U.S. Pat. No. 5,672,686, which is a continuation-in-part of U.S. application Ser. No. 08/287,427, filed Aug. 9, 1994, now abandoned, (bak is referred to therein as bcl-y), the disclosures of which are incorporated herein by reference. Ectopic Bak expression accelerates the death of an IL-3 dependent cell line upon cytokine withdrawal, and opposes the protection against apoptosis afforded by Bcl-2. In addition, enforced expression of Bak is sufficient to induce apoptosis of serum deprived fibroblasts, raising the possibility that Bak directly activates, or is itself a component of, the cell death machinery.
Known cellular Bcl-2 related genes, where analyzed, have distinct patterns of expression and thus may function in different tissues. The cell death program is in place in all tissues and may be regulated by different Bcl-2 related genes. While Bcl-2 expression is required for maintenance of the mature immune system, it is desirable to identify other genes which may govern apoptotic cell death in other lineages. From the perspective of pharmaceutical development, it would be desirable to identify or develop agents that either activate or suppress apoptosis, depending on the clinical setting.